Data collection, management and analysis

Participants will be identified by study ID. Study data are collected and managed using the paper case report forms. Laboratory data will be transferred electronically from the laboratory performing clinical analyses and will be archived in secured hard drives with backup. All biological material (blood, tongue tissue) obtained from the study participants will be kept in a research bio-bank. Samples will be labelled with study ID. The bio-bank allows for the analyses of samples to be performed simultaneously to avoid large instrumental variations. The trial adheres to the Data Protection Act which requires data to be anonymized as soon as it is practical to do so.

Analyses will be based on two defined analysis sets: an intention-to-treat (ITT) analysis set and a per-protocol (PP) analysis set. ITT includes all randomized participants exposed to at least one dose of trial product that have completed the visit at week 2. The per-protocol population will include all patients completing the study with a documented valid baseline and end of treatment assessment of the primary endpoint without a major protocol violation. Safety analysis will be performed on the ITT analysis set. Two-tailed tests will be performed and the significance level will be set to α = 0.05.

All information concerning the subjects will be recorded and saved on password-protected computers. A detailed database will be set up to track each subject’s progress through this trial, including the scheduling of baseline and endpoint assessments. All the generated data will be stored on password-protected computers or servers that are only accessible to the research team.

Data and resources will be shared with other eligible investigators through academically established means. The protocol and datasets used and/or analysed in this study will be available from the corresponding author on reasonable request. Researchers who provide a methodologically sound proposal may access data to achieve aims in the approved proposal.

The non-parametric Mann-Whitney test or Kruskal-Wallis test was used to compare the distribution of continuous variables between the different groups. The Wilcoxon signed-rank test was used for the comparison of continuous variables for related samples. Spearman’s rho (ρ) was used to assess the correlation between continuous variables. P values below 0.05 were considered statistically significant. Statistical analysis was performed using IBM SPSS Statistics, version 27.0 (IBM Corporation, Armonk, NY, USA).

The stability of differential gene expression will be controlled by cross-validation analysis of RNA-seq data. Differential expression (DE) between the groups will be analysed by application of edgeR and DESeq2 tools [29, 30]. Positive results will be controlled for false discovery rate (FDR) by the adaptive method of Benjamini et al. [31]. A list of differentially expressed genes with at least 2-fold difference in expression, called by both DE tools and passing the FDR test, will be collected, and Gene Ontology enrichment analysis will be performed to identify the potential signalling pathways and cellular processes of significance [32].